Coronary artery and peripheral vascular angiemphraxis is the most fatal factor for human health. Intravascular stenting has become one of the most effective therapy methods to release such diseases easily. After implantation, the first generation stent, bare metal stent, can cause inflammation, in the long stretch leading to intravascular intimal hyperplasia, and further stimulate the growth factor and cytokine secretion, leading to smooth muscle cell proliferation and migration, caused in-stent restenosis which occurs in about 15 to 30% of the procedures. The second generation drug eluting stent, such as rapamycin or paclitaxel stents, have dramatically reduced the incidence of in-stent restenosis and the incidence of adverse events. Anti-proliferation drug, interfere with the natural healing response by preventing or significantly delaying the formation of a functional endothelial lining over the stent, increasing the risk of the late in-stent thrombosis. Most drug-eluting stents use synthetic polymer matrices as coatings. Increasing evidence suggests that some adverse reactions, such as hypersensitivity reactions, inflammatory reactions and vascular intimal hyperplasia have been occurred and clinical therapy results suggest that drug-eluting stent might casuse late restenosis after operation. Drug coatings can inhibit the proliferation of smooth muscle cells and the regeneration of ECs, delaying the endothelialization of blood vessel and increasing the risk of late thrombosis. As shown in studies that using bare metal stent does not influence the physiological reaction of proximate and distal blood vessel of stent, but using drug eluting metal stents lead to paradoxical contraction of proximate and distal blood vessel of stent. The findings indicate that the diffusion of anti-proliferation drug on drug-eluting stents may cause injury of blood vessel endothelium, and may also be the cause of paradoxical reaction of blood vessel. The FDA reported 50 hypersensitivity reactions after stent placement for 6 months, such as tetter, dyspnoea, urticaria, pruritus, and febricity. The reports and autopsy findings suggest that systemic hypersensitivity reactions that, in some cases, have been associated with late thrombosis and death. An important uncertain factor in the efficacy of drug-eluting stent is the use of polymers.
In 1997, Asahara isolated vascular endothelial progenitor cells (EPCs) from human peripheral blood with anti-CD34 and anti-vascular endothelial growth factor 2 (VEGFR-2). In vitro, these cells differentiated into functional vascular endothelial cells (ECs). The in vivo results suggested that EPCs may contribute to neoangiogenesis in adult species, consistent with vasculogenesis, a paradigm otherwise restricted to embryogenesis[Science, 1997, 275 (5320):964]. In 2003, Toshihiko et al. have shown accelerated endothelialization on polyethylene terephthalate stent preclotted with autologous bone marrow cells having a subset of early ECs that express the CD34 antigen on their surfaces. The authors used composite stent implanted in the canine's descending thoracic aorta and carotid artery for 4 weeks. The study stent was treated with CD34+ bone marrow cells mixed with venous blood; the control stent was treated with ECs mixed with venous blood only. Histologic evaluation in one week demonstrated significant increases of surface endothelialization on the seeded stents (92%±3.4% vs 26.6%±7.6%) compared with controls. Four weeks later, on the seeded stents, there was a layer of neointima consisting of a single layer of ECs shown to be positive with VEGFR-2 and CD34+ staining on the surface. Most of the control stent surfaces were covered with a thin layer of pseudointima. There were no ECs on the pseudointima, which was largely composed of a fibrin coagulum with some red cells, macrophages, neutrophils, giant cells, and occasional α-actin positive cells. [Biomaterials 2003, 24:2295]
In 2005, Kutryk et al designed CD34 antibody (CD34) stent with the technology of isolating EPCs from human blood by magnetic bead selection on the basis of cell surface antigen expression. CD34 were immobilized to the stent surface with PTFE. [U.S. Pat. No. 7,037,332] The CD34 stent have been shown to exhibit cross-reactivity in porcine stent explants, which were observed to have a rich population of EPCs after only 48 hours. The third generation antibody coating stents have been developed using immobilized antibodies targeted at EPCs surface antigens. The early establishment of a functional endothelial layer after vascular injury has been shown to assist in the prevention of neointimal proliferation and thrombus formation. These preclinical and preliminary clinical results have to be interpreted carefully, considering the recent emergence of new technologies such as drug-eluting stent. Drug-eluting stent inhibit the inflammatory and proliferative process of the normal healing response, including the formation of a confluent endothelial layer on the stent. The EPCs capture stent induced the rapid establishment of a functional endothelial layer early in the healing response. [J. American College of Cardiology 2005, 45(10):1574] However, the CD34 carrier coating materials are synthetic fat-soluble materials such as PTFE or polyurethane. CD34 is immunoglobulin IgG1. PTFE was not well compatible with CD34. Stent coating was prepared by immersing the stent into PTFE tetrahydrofuran solution mixed with CD34, dipping to the surface of stent after emulsification. After drying, the natural state of protein secondary structure of CD34 gradually changed under the anhydrous environment. Therefore, this CD34 stent also can not have desirable biologic stability in a long period. Kutryk et al designed that CD34 was chemically crosslinked onto functional matrix coating on the metal stent. The major problem of chemical crosslinking is that partical effective sites of antibody are chemically crosslinked. The activity of CD34 is going to lose gradually without water in the coating. In addition, CD34 lacks specificity on EPCs, so the coating can adsorb EPCs and ECs which CD34+ at the same time. The PTFE matrix can not provide a suitable place for the differentiation of vascular ECs due to its poor biocompatibility. [Criculation Jul. 5, 2005, 12-17]
In 1997, Sheri Miraglia et al described the production of CD133 monoclonal antibody (CD133). CD133+ binds to a novel cell surface antigen present on CD34+ bright subset of human hematopoietic stem and progenitor cells, suggesting that it may be an important early marker for hematopoietic stem and progenitor cells. [Blood 90: 5002-5012; 5013-5021] Wang Mingyuan showed that in the process of CD34+/CD133+ EPCs differentiating into mature ECs in vitro, stem cell markers CD133+ has been gradually declining, which indicates that EPCs is in a transition phenotype stage from the blood stem cells to ECs. With the processes of differentiation and maturation, cell phenotypes are gradually changed by some gene regulation. CD34+ is the most common hematopoietic stem cell marker, expressing ECs line. CD133+ is a newly discovered stem cell marker, not expressing on mature ECs that distinction is the vascular ECs and EPCs the only marker. Therefore CD133+ is an earlier marker for expressing HSPCs than CD34+. HSPCs of CD133+/CD34+ and CD133−/CD34+ expressed stem cell (Stem cell expressed by HSPCs of CD133+/CD34+ and CD133−/CD34+) are about 0.080% and 0.034% in adult peripheral blood, respectively. CD34+ is expressed by EPCs, circulating ECs, common myeloid progenitor (myelomonocytic precursors, megakaryocytic/erythroid precursors) and common lymphoid progenitor. CD133+ is a more specific marker for expressing EPCs. In other mature blood cells, such as nucleated red blood cells, lymphocytes, myelocytic, mononuclear and platelets, CD133+ expressions were not detected. In other types of haemopoietic stem cells, CD133+ expression was not detected. Therefore, VEGFR-2 or CD34 is not the ideal choice for the EPCs capture, but CD133 is the more specific choice. [J. Clin Invest, 2002, 109:337]
The amidoglucosan polysaccharides in nature include basic amidoglucosan polysaccharides and acidic amidoglucosan polysaccharides, which can be degraded gradually by lysozyme in vivo. Chitosan (CH) is a linear polysaccharide, containing two β-1,4-linked 2-amino-2-deoxy-D-glucopyranose, obtained by partial de-N-acetylation of chitin, which has good biocompatibility, biodegradability and antimicrobality. CH has been approved by FDA to use as biodegradable surgical suture material. It can inhibit vascular smooth muscle cell proliferation, promote the growth of ECs and improve the wound healing. All the properties of CH described above demonstrated that CH can be applied in the field of stent coating material so as to prevent restenosis. [Acad. J. Sec. Mil. Med. Univ. 20 (1999) 962] In addition, carboxymethyl-chitosan is acidic amidoglucosan polysaccharide from carboxymethylation of CH with good biocompatibility, water retention capacity, flexibility, washing resestance, biological stability [Acad. J. Sec. Mil. Med. Univ. 15 (1994):452] There are basically six acidic amidoglucosan polysaccharides in nature, such as hyaluronic acid, heparin, chondroitin sulfate, dermatan sulfate, keratin sulfate. Hyaluronic acid (HA) is a ubiquitous component of extracellular matrix. HA is a linear polysaccharide containing two β-1,4-linked 2-amino-2-deoxy-D-glucopyranose. HA is a very important glycosamineglycan in human tissue. It has become an important medical biopolymer material and has been widely used in medical bioengineering. In 2005, HA as wound dressing was approved by FDA. [Primaphamr] HA shows high affinity for injured tissue to provide suitable environment for cell proliferation and differentiation, promoting cell growth, differentiation, reconstruction and rehabilitation. Especially HA can promote ECs proliferation and angiogenesis in blood vessel of human and mammalian as coating of endovascular devices. HA has been shown to inhibit platelet aggregation and adhesion and to prolong the bleeding time. Because of its antithrombotic effects and its known coating abilities, HA may provide a potential biocompatible and thromboresistant coating for endovascular devices to anticoagulated blood under arterial blood flow conditions. These properties make HA an excellent material for fabricating stent coatings to provide an artificial extracellular matrix environment suitable for encapsulated cells differentiation to prevent restenosis. [J. Biomed Mater Res, 2000, 05:101-109; International Congress Series, 2001, 1223:2279-2284; Biomacromolecules 2003, 4:1564-1571]
The ideal stent coatings should have good biocompatibility, accelerating promoting injured tissue healing, preventing excessive proliferation, accelerating vascular endothelium, preventing thrombosis and restenosis, and also should have biological stability. In this invention, layer-by-layer self-assembly two polysaccharides, HA and CH, were employed to multilayer coating loading with CD133 for endovascular stent. After implanting this stent in vessel, EPCs of the peripheral blood can be captured specifically by CD133, then differentiated into ECs. Amidoglucosan polysaccharides have good biocompatibility, which provides suitable location for the differentiation of EPCs into ECs and monolayer ECs overburden layer on stent can form in 48 h, which can effectively avoid the formation of partial “pseudointima”. This would repair accelerative vessel injury caused by stent expanding and would be a more natural way to prevent restenosis and thrombosis.